5.2 INOCULUM PREPARATION:

5.2.1 Inoculate 1 ml of the frozen culture of both organism P.aeruginosa-ATCC 9027 and E.Coli-ATCC-8739 from cryovial to 2 x 20 ml (Two tubes for each) tubes containing soybean casein digest medium. Incubate all tubes at 30°C-35°C for 24 hrs.5.2.2 Spread one ml of growth culture from each tube on a separate plate containing SCD agar. Incubate at 30°C-35°C for 24 hrs.5.2.3 Add 5 ml 0.9% sterile saline in one of the plates of each organism.5.2.4 Collect the growth in a sterile test tube with the help of sterile pipette.5.2.5 Prepare serial dilution from it & take the viable count from each dilution in duplicate.5.2.6 Prepare suspension of above two microorganisms to get final concentration 10^6 cfu/ml. Record the result in Annexure-II Part A.5.3 PRE INCUBATION OF MEDIA FILLED VIAL:Pre-incubate the media filled and sealed vial at 30-35°C for about 2-3days to ensure that the vials are free from contamination.5.4 SAMPLING QUANTITY: (20 vials for each organism)

5.5 PROCEDURE

5.5.1 Place vials in two different beakers in inverted position & then pour a sufficient quantity of each suspension respectively such that the closure and vial neck are covered completely.5.5.2 Keep it for 30 min.5.5.3 Slowly take out the vials one by one & place it on the stand in inverted position. For two different organisms, use separate stand..5.5.4 Cover the stand of both organisms with a plastic bag to avoid outside contamination.5.5.5 Incubate at 30°C-35°C for 14 days.5.5.6 Take a viable count of both suspension before and after testing & record the result in Annexure-II. Part B & C.5.5.7 If any units show growth investigate whether it comes from the microorganism used for the test or due to contamination.5.5.8 Records the result in Annexure-I.

5.6 GROWTH PROMOTION TEST:

5.6.1 After completion of 14 days, do the growth promotion test using both microorganisms in two to three vials for each microorganism.5.6.2 Record the results in Annexure-I.